Bifunctional compounds which link cytotoxic reagents to antibodies (i.e., xe2x80x9clinkersxe2x80x9d) are known in the art. These compounds have been particularly useful in the formation of immunoconjugates directed against tumor associated antigens. Such immunoconjugates allow the selective delivery of toxic drugs to tumor cells. (See e.g., Hermentin and Seiler, xe2x80x9cInvestigations With Monoclonal Antibody Drug Conjugates,xe2x80x9d Behringer Insti. Mitl. 82:197-215 (1988); Gallego et al., xe2x80x9cPreparation of Four Daunomycin-Monoclonal Antibody 791T/36 Conjugates With Anti-Tumor Activity,xe2x80x9d Int. J. Cancer 33:7737-44 (1984); Arnon et al., xe2x80x9cIn Vitro and In Vivo Efficacy of Conjugates of Daunomycin With Anti-Tumor Antibodies,xe2x80x9d Immunological Rev. 62:5-27 (1982).
Greenfield et al. have described the formation of acid-sensitive immunoconjugates containing the acylhydrazine compound, 3-(2-pyridyl-dithio)propionyl hydrazide conjugated via an acylhydrazone bond to the 13-keto position of an anthracycline molecule, and conjugation of this anthracycline derivative to an antibody molecule (Greenfield et al., European Patent Publication EP 0 328 147, published Aug. 16, 1989, which corresponds to pending U.S. Ser. No. 07/270,509, filed Nov. 16, 1988, now abandoned and U.S. Ser. No. 07/155,181, filed Feb. 11, 1988, now abandoned). This latter reference also discloses specific thioether-containing linkers and conjugates, including hydrazone thioether containing immunoconjugates.
Kaneko et al. (U.S. Ser. No. 07/522,996, filed May 14, 1990, now U.S. Pat. No. 5,137,877 which is equivalent to European Patent Publication, EP A 0 457 250, published Nov. 21, 1991) have also described the formation of conjugates containing anthracycline antibiotics attached to a bifunctional linker by an acylhydrazone bond at the C-13 position of an anthracycline molecule. In their invention the linkers contain a reactive pyridinyldithio- or an ortho-nitrophenyldithio-group, by which the linker reacts with a suitable group attached to a cell reactive ligand, to form the completed conjugate. An important consideration for immunoconjugates is that the relationship between drug potency and antigen expression must be appropriate in order to effect cytotoxicity on a broad range of malignant cells. Alterations in the potency of various immunoconjugates can be affected by changing the monoclonal antibody (MAb) utilized and/or the potency of the unconjugated drug. It is also possible to effect the potency of immunoconjugates by changes in the linker, both in terms of stability in circulation (Koizumi, M., K., Kunimatsu, M., Sakahara, H., Nakashima, T., Kawamura, Y., Watanabe, Y., Ohmomo, Y., Arano, Y., Yokoyama, A. and Torizuka, K. (1987), Preparation of 67Ga-labeled antibodies using deferoxamine as a bifunctional chelate. J. Immunol Methods 104, 93-102; Thorpe, P. E., Wallace, P. M., Knowless, P. P., Relf, M G., Brown, A. N. F., Watson, G. J. Knyba, R. E., Wawrzynczak, E. J. and Blakey, D. C. (1987), New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo. Cancer Res. 47,5924-5931; Trail, P. A., Wilner, D., Lasch, S. J., Henderson, A. J., Greenfield, R. S., King, D., Zoeckler, M. E. and Braslawsky, G. R. (1992), Antigen specific activity of carcinoma reactive BR64-adriamycin conjugates evaluated in vitro and in human tumor xenograft modelsk, Cancer Research 52, 5693-5700; Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone R. A., Hellstrxc3x6m, K. E. (1993), Cure of xenografted human carcinomas by BR96-Doxorubicin Immuno-conjugates, Science 261, 212-215; Trail, P. A., Willner, D. and Hellstrom, K. E. (1995), Site-directed delivery of anthracyclines for cancer therapy. Drug Development Research 34, 196-209) and in terms of drug/MAb molar ratio (Shih, L. B., Goldenberg, D. M., Xuan, H., Lu, H., Sharkey, R. M. and Hall, T. C. (1991), Anthracycline immunoconjugates prepared by a site specific linkage via an aminodextran intermediate carrier. International Journal of Cancer 41, 8320839; Trail et al.1992; Trail et al.,1995).
In particular, the in vitro potency of doxorubicin conjugates prepared with the internalizing anticarcinoma MAb BR64 and an acid labile hydrazone bond, was shown to increase as drug/MAb molar ratios increased from 1-8 (Trail et al.,1992; Trail et al.,1995). However, in these studies the increase in drug/MAb molar ratios was based on increasing the number of conjugation sites on the MAb which is self-limiting and has other drawbacks such as reduced antibody binding affinity.
In view of the above, it is clear that one of the problems in prior art immunoconjugates is the relatively low ratio of drug to targeting ligand (e.g., immunoglobulin) achievable. It would be highly desirable to have immunoconjugates which provide a higher ratio of drug to targeting ligand.
The present invention provides novel branched hydrazone linkers. The novel linkers are used to prepare novel drug/linker molecules and biologically active conjugates composed of a targeting ligand, a therapeutically active drug , and a branched linker capable of recognizing a selected target cell population (e.g., tumor cells) via the targeting ligand.
As used herein the term xe2x80x9cdrug/linkerxe2x80x9d or xe2x80x9clinker/drugxe2x80x9d molecule refers to the linker molecule coupled to two or more therapeutically active drug molecules, and the term xe2x80x9cconjugatexe2x80x9d refers to the drug/linker molecule coupled to the targeting ligand. The linkers are branched so that more than one drug molecule per linker are coupled to the ligand. The number of drugs attached to each linker varies by a factor of 2 for each generation of branching. Thus, the number of drug molecules per molecule of linker can be 2, 4, 8, 16, 32, 64, etc. The factor of branching can be expressed mathematically as 2n wherein n is a positive integer. Thus, a singly branched linker will have a first generation of branching or 21, i.e., contains two drug molecules per linker. A doubly branched linker will have a second generation of branching or 22, i.e., contains four drug molecules per linker.
Thus, the present invention is directed to a branched linker for linking a thiol group derived from a targeting ligand to two or more drug moieties which comprises a compound having a terminus containing a thiol acceptor for binding to a thiol group (also called a sulfhydryl group) derived from a targeting ligand, at least one point of branching which is a polyvalent atom allowing for a level of branching of 2n wherein n is a positive integer, and at least two other termini containing acylhydrazide groups capable of forming acylhyrdazone bonds with aldehyde or keto groups derived from a drug moiety. It is preferred that n is 1,2, 3, or 4; more preferably 1, 2 or 3; most preferably 1 or 2. It is also preferred that the polyvalent atom is carbon or nitrogen, and the targeting ligand is an antibody or fragment thereof.
As used in the preceeding paragraph, the phrase xe2x80x9cthiol group derived from the targeting ligandxe2x80x9d means that the thiol group is already present on the targeting ligand or that the targeting ligand is chemically modified to contain a thiol group, which modification optionally includes a thiol spacer group between the targeting ligand and the thiol group. Likewise, the phrase xe2x80x9can aldehyde or keto group derived from a drug moietyxe2x80x9d means that the aldehyde or keto group is already present on the drug or the drug is chemically modified to contain an aldehyde or keto group.
Also provided by the invention are intermediates for preparing the linkers, drug/linkers and/or conjugates; and a method for treating or preventing a selected disease state which comprises administering to a patient a conjugate of the invention.